Packaging systems for human recombinant adenovirus to be used in gene therapy

ABSTRACT

Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector are suited for large-scale production of recombinant adenoviruses. A system for use with the invention produces replication-defective adenovirus. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, has at least one functional encapsidation signal and at least one functional Inverted Terminal Repeat, and lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of Ser. No. 09/918,029, filedJul. 30, 2001, now U.S. Pat. No. 6,783,980, which is a divisional ofSer. No. 09/506,548 filed Feb. 16, 2000, now U.S. Pat. No. 6,602,706,which is a divisional of Ser. No. 09/334,765 filed Jun. 16, 1999, nowU.S. Pat. No. 6,238,893, which is a continuation of Ser. No. 08/793,170filed Mar. 25, 1997, now U.S. Pat. No. 5,994,128, which is the nationalstage of International Patent Application PCT/NL96/00244 filed on Jun.14, 1996, which itself claims priority from European patent application95201728.3 filed on Jun. 26, 1995, and European patent application95201611.1 filed on Jun. 15, 1995, each of which is incorporated hereinin its entirety by this reference.

TECHNICAL FIELD

The invention relates to the field of recombinant DNA technology, morein particular to the field of gene therapy. In particular, the inventionrelates to gene therapy using materials derived from adenovirus,specifically human recombinant adenovirus. It especially relates tonovel virus-derived vectors and novel packaging cell lines for vectorsbased on adenoviruses.

BACKGROUND

Gene therapy is a recently developed concept for which a wide range ofapplications can be and have been envisioned. In gene therapy, amolecule carrying genetic information is introduced into some or allcells of a host, as a result of which the genetic information is addedto the host in a functional format.

The genetic information added may be a gene or a derivative of a gene,such as a cDNA, which encodes a protein. This is a functional format inthat the protein can be expressed by the machinery of the host cell.

The genetic information can also be a sequence of nucleotidescomplementary to a sequence of nucleotides (either DNA or RNA) presentin the host cell. This is a functional format in that the added DNA(nucleic acid) molecule or copies made thereof in situ are capable ofbase pairing with the complementary sequence present in the host cell.

Applications include the treatment of genetic disorders by supplementinga protein or other substance which, because of the genetic disorder, iseither absent or present in insufficient amounts in the host, thetreatment of tumors, and the treatment of other acquired diseases suchas (auto)immune diseases, infections, etc.

As may be inferred from the above, there are basically three differentapproaches in gene therapy: the first directed towards compensating fora deficiency in a (mammalian) host, the second directed towards theremoval or elimination of unwanted substances (organisms or cells) andthe third towards application of a recombinant vaccine (against tumorsor foreign microorganisms).

For the purpose of gene therapy, adenoviruses carrying deletions havebeen proposed as suitable vehicles for genetic information. Adenovirusesare nonenveloped DNA viruses. Gene-transfer vectors derived fromadenoviruses (so-called “adenoviral vectors”) have a number of featuresthat make them particularly useful for gene transfer for such purposes.For example, the biology of the adenovirus is characterized in detail,the adenovirus is not associated with severe human pathology, theadenovirus is extremely efficient in introducing its DNA into the hostcell, the adenovirus can infect a wide variety of cells and has a broadhost-range, the adenovirus can be produced in large quantities withrelative ease, and the adenovirus can be rendered replication defectiveby deletions in the early-region 1 (“E1”) of the viral genome.

The adenovirus genome is a linear double-stranded DNA molecule ofapproximately 36,000 base pairs with the 55 kDa terminal proteincovalently bound to the 5′ terminus of each strand. The adenoviral(“Ad”) DNA contains identical Inverted Terminal Repeats (“ITR”) of about100 base pairs with the exact length depending on the serotype. Theviral origins of replication are located within the ITRs exactly at thegenome ends. DNA synthesis occurs in two stages. First, the replicationproceeds by strand displacement, generating a daughter duplex moleculeand a parental displaced strand. The displaced strand is single strandedand can form a so-called “panhandle” intermediate, which allowsreplication initiation and generation of a daughter duplex molecule.Alternatively, replication may proceed from both ends of the genomesimultaneously, obviating the requirement to form the panhandlestructure. The replication is summarized in FIG. 14 adapted from Lechnerand Kelly (1977).

During the productive infection cycle, the viral genes are expressed intwo phases: the early phase, which is the period up to viral DNAreplication, and the late phase, which coincides with the initiation ofviral DNA replication. During the early phase, only the early geneproducts, encoded by regions E1, E2, E3 and E4, are expressed, whichcarry out a number of functions that prepare the cell for synthesis ofviral structural proteins (Berk, 1986). During the late phase, the lateviral gene products are expressed in addition to the early geneproducts, and host cell DNA and protein synthesis are shut off.Consequently, the cell becomes dedicated to the production of viral DNAand of viral structural proteins (Tooze, 1981).

The E1 region of adenovirus is the first region of adenovirus expressedafter infection of the target cell. This region consists of twotranscriptional units, the E1A and E1B genes, which both are requiredfor oncogenic transformation of primary (embryonic) rodent cultures. Themain functions of the E1A gene products are 1) to induce quiescent cellsto enter the cell cycle and resume cellular DNA synthesis and 2) totranscriptionally activate the E1B gene and the other early regions (E2,E3, E4). Transfection of primary cells with the E1A gene alone caninduce unlimited proliferation (immortalization) but does not result incomplete transformation. However, expression of E1A in most casesresults in induction of programmed cell death (apoptosis) and onlyoccasionally immortalization (Jochemsen et al., 1987). Co-expression ofthe E1B gene is required to prevent induction of apoptosis and forcomplete morphological transformation to occur. In established immortalcell lines, high level expression of E1A can cause completetransformation in the absence of E1B (Roberts et al., 1985).

The E1B encoded proteins assist E1A in redirecting the cellularfunctions to allow viral replication. The E1B 55 kDa and E4 33 kDaproteins, which form a complex that is essentially localized in thenucleus, function in inhibiting the synthesis of host proteins and infacilitating the expression of viral genes. Their main influence is toestablish selective transport of viral mRNAs from the nucleus to thecytoplasm concomitantly with the onset of the late phase of infection.The E1B 21 kDa protein is important for correct temporal control of theproductive infection cycle, thereby preventing premature death of thehost cell before the virus life cycle has been completed. Mutant virusesincapable of expressing the E1B 21 kDa gene product exhibit a shortenedinfection cycle that is accompanied by excessive degradation of hostcell chromosomal DNA (deg-phenotype) and in an enhanced cytopathiceffect (cyt-phenotype) (Telling et al., 1994). The deg and cytphenotypes are suppressed when, in addition, the E1A gene is mutated,indicating that these phenotypes are a function of E1A (White et al.,1988). Furthermore, the E1B 21 kDa protein slows down the rate by whichE1A switches on the other viral genes. It is not yet known through whichmechanisms E1B 21 kDa quenches these E1A-dependent functions.

Vectors derived from human adenoviruses, in which at least the E1 regionhas been deleted and replaced by a gene of interest, have been usedextensively for gene therapy experiments in the preclinical and clinicalphase.

As stated before, all adenovirus vectors currently used in gene therapyare believed to have a deletion in the E1 region, where novel geneticinformation can be introduced. The E1 deletion renders the recombinantvirus replication defective (Stratford-Perricaudet and Perricaudet,1991). We have demonstrated that recombinant adenoviruses are able toefficiently transfer recombinant genes to the rat liver and airwayepithelium of rhesus monkeys (Bout et al., 1994b; Bout et al., 1994a).In addition, we (Vincent et al., 1996a; Vincent et al., 1996b) andothers (see, e.g., Haddada et al., 1993) have observed a very efficientin vivo adenovirus mediated gene transfer to a variety of tumor cells invitro and to solid tumors in animal models (lung tumors, glioma) andhuman xenografts in immunodeficient mice (lung) in vivo (reviewed byBlaese et al., 1995).

In contrast to (for instance) retroviruses, adenoviruses 1) do notintegrate into the host cell genome, 2) are able to infect nondividingcells, and 3) are able to efficiently transfer recombinant genes in vivo(Brody and Crystal, 1994). Those features make adenoviruses attractivecandidates for in vivo gene transfer of, for instance, suicide orcytokine genes into tumor cells.

However, a problem associated with current recombinant adenovirustechnology is the possibility of unwanted generation of replicationcompetent adenovirus (“RCA”) during the production of recombinantadenovirus (Lochmüller et al., 1994; Imler et al., 1996). This is causedby homologous recombination between overlapping sequences from therecombinant vector and the adenovirus constructs present in thecomplementing cell line, such as the 293 cells (Graham et al., 1977).RCA is undesirable in batches to be used in clinical trials becauseRCA 1) will replicate in an uncontrolled fashion, 2) can complementreplication-defective recombinant adenovirus, causing uncontrolledmultiplication of the recombinant adenovirus, and 3) batches containingRCA induce significant tissue damage and hence strong pathological sideeffects (Lochmüller et al., 1994). Therefore, batches to be used inclinical trials should be proven free of RCA (Ostrove, 1994).

It was generally thought that E1-deleted vectors would not express anyother adenovirus genes. However, recently it has been demonstrated thatsome cell types are able to express adenovirus genes in the absence ofE1 sequences. This indicates that some cell types possess the machineryto drive transcription of adenovirus genes. In particular, it wasdemonstrated that such cells synthesize E2A and late adenovirusproteins.

In a gene therapy setting, this means that transfer of the therapeuticrecombinant gene to somatic cells not only results in expression of thetherapeutic protein but may also result in the synthesis of viralproteins. Cells that express adenoviral proteins are recognized andkilled by Cytotoxic T Lymphocytes, which thus 1) eradicates thetransduced cells and 2) causes inflammations (Bout et al., 1994a;Engelhardt et al., 1993; Simon et al., 1993). As this adverse reactionhampers gene therapy, several solutions to this problem have beensuggested, such as 1) using immunosuppressive agents after treatment, 2)retention of the adenovirus E3 region in the recombinant vector (seeEuropean patent application EP 95202213), and 3) usingtemperature-sensitive (“ts”) mutants of human adenovirus, which have apoint mutation in the E2A region rendering them temperature sensitive,as has been claimed in patent WO/28938.

However, these strategies to circumvent the immune response have theirlimitations. The use of ts mutant recombinant adenovirus diminishes theimmune response to some extent but was less effective in preventingpathological responses in the lungs (Engelhardt et al., 1994a).

The E2A protein may induce an immune response by itself, and it plays apivotal role in the switch to the synthesis of late adenovirus proteins.Therefore, it is attractive to make temperature-sensitive recombinanthuman adenoviruses.

A major drawback of this system is the fact that although the E2 proteinis unstable at the nonpermissive temperature, the immunogenic protein isstill being synthesized. In addition, it is to be expected that theunstable protein does activate late gene expression, albeit to a lowextent. ts125 mutant recombinant adenoviruses have been tested, andprolonged recombinant gene expression was reported (Yang et al., 1994b;Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al., 1995).However, pathology in the lungs of cotton rats was still high(Engelhardt et al., 1994a), indicating that the use of ts mutantsresults in only a partial improvement in recombinant adenovirustechnology. Others (Fang et al., 1996) did not observe prolonged geneexpression in mice and dogs using ts125 recombinant adenovirus. Anadditional difficulty associated with the use of ts125 mutantadenoviruses is that a high frequency of reversion is observed. Theserevertants are either real revertants or the result of second sitemutations (Kruijer et al., 1983; Nicolas et al., 1981). Both types ofrevertants have an E2A protein that functions at normal temperature and,therefore, have toxicity similar to the wild-type virus.

SUMMARY OF THE INVENTION

In one aspect of the invention, this problem in virus production issolved in that we have developed packaging cells that have nooverlapping sequences with a new basic vector and thus are suited forsafe large-scale production of recombinant adenoviruses. One of theadditional problems associated with the use of recombinant adenovirusvectors is the host-defense reaction against treatment with adenovirus.

Briefly, recombinant adenoviruses are deleted for the E1 region. Theadenovirus E1 products trigger the transcription of the other earlygenes (E2, E3, E4), which consequently activate expression of the latevirus genes.

In another aspect of the present invention we, therefore, delete E2Acoding sequences from the recombinant adenovirus genome and transfectthese E2A sequences into the (packaging) cell lines containing E1sequences to complement recombinant adenovirus vectors.

Major hurdles in this approach are 1) that E2A should be expressed tovery high levels and 2) that E2A protein is very toxic to cells.

The current invention, in yet another aspect, therefore, discloses useof the ts125 mutant E2A gene, which produces a protein that is not ableto bind DNA sequences at the non-permissive temperature. High levels ofthis protein may be maintained in the cells (because it is nontoxic atthis temperature) until the switch to the permissive temperature ismade. This can be combined with placing the mutant E2A gene under thedirection of an inducible promoter, such as, tet, methallothionein,steroid inducible promoter, retinoic acid βZreceptor, or other induciblesystems. However, in yet another aspect of the invention, the use of aninducible promoter to control the moment of production of toxicwild-type E2A is disclosed.

Two salient additional advantages of E2A-deleted recombinant adenovirusare 1) the increased capacity to harbor heterologous sequences and 2)the permanent selection for cells that express the mutant E2A. Thissecond advantage relates to the high frequency of reversion of ts125mutation: when reversion occurs in a cell line harboring ts125 E2A, thiswill be lethal to the cell. Therefore, there is a permanent selectionfor those cells that express the ts125 mutant E2A protein. In addition,as we, in one aspect of the invention, generate E2A-deleted recombinantadenovirus, we will not have the problem of reversion in ouradenoviruses.

In yet another aspect of the invention, as a further improvement, theuse of nonhuman cell lines as packaging cell lines is disclosed.

For GMP production of clinical batches of recombinant viruses, it isdesirable to use a cell line that has been used widely for production ofother biotechnology products. Most of the latter cell lines are ofmonkey origin, which have been used to produce, for example, vaccines.

These cells cannot be used directly for the production of recombinanthuman adenovirus, as human adenovirus cannot replicate or replicatesonly to low levels in cells of monkey origin. A block in the switch ofthe early to late phase of the adenovirus lytic cycle underliesdefective replication. However, host range mutations in the humanadenovirus genome are described (hr400–404), which allow replication ofhuman viruses in monkey cells. These mutations reside in the geneencoding E2A protein (Klessig and Grodzicker, 1979; Klessig et al.,1984; Rice and Klessig, 1985). Moreover, mutant viruses have beendescribed that harbor both the hr and temperature-sensitive ts125phenotype (Brough et al., 1985; Rice and Klessig, 1985).

We, therefore, generate packaging cell lines of monkey origin (e.g.,VERO, CV1) that harbor:

-   -   1) E1 sequences, to allow replication of E1/E2-defective        adenoviruses, and    -   2) E2A sequences, containing the hr mutation and the ts125        mutation, named ts400 (Brough et al., 1985; Rice and        Klessig, 1985) to prevent cell death by E2A overexpression,        and/or    -   3) E2A sequences, just containing the hr mutation, under the        control of an inducible promoter, and/or    -   4) E2A sequences, containing the hr mutation and the ts125        mutation (ts400), under the control of an inducible promoter.

Furthermore, we disclose the construction of novel and improvedcombinations of packaging cell lines and recombinant adenovirus vectors.We provide:

-   -   1) A novel packaging cell line derived from diploid human        embryonic retinoblasts (“HER”) that harbors nt. 80–5788 of the        Ad5 genome. This cell line, named 911, deposited under        no.95062101 at the ECACC™, has many characteristics that make it        superior to the commonly used 293 cells (Fallaux et al., 1996);    -   2) Novel packaging cell lines that express just E1A genes and        not E1B genes. Established cell lines (and not human diploid        cells of which 293 and 911 cells are derived) are able to        express E1A to high levels without undergoing apoptotic cell        death, as occurs in human diploid cells that express E1A in the        absence of E1B. Such cell lines are able to trans-complement        E1B-defective recombinant adenoviruses, because viruses mutated        for E1B 21 kDa protein are able to complete viral replication        even faster than wild-type adenoviruses (Telling et al., 1994).        The constructs are described in detail below and are graphically        represented in FIGS. 1–5. The constructs are transfected into        the different established cell lines and are selected for high        expression of E1A. This is done by operatively linking a        selectable marker gene (e.g., NEO gene) directly to the E1B        promoter. The E1B promoter is transcriptionally activated by the        E1A gene product and, therefore, resistance to the selective        agent (e.g., G418 in the case of NEO is used as the selection        marker) results in direct selection for desired expression of        the E1A gene;    -   3) Packaging constructs that are mutated or deleted for E1B 21        kDa, but just express the 55 kDa protein;    -   4) Packaging constructs to be used for generation of        complementing packaging cell lines from diploid cells (not        exclusively of human origin) without the need for selection with        marker genes. These cells are immortalized by expression of E1A.        However, in this particular case, expression of E1B is essential        to prevent apoptosis induced by E1A proteins. Selection of        E1-expressing cells is achieved by selection for focus formation        (immortalization), as described for 293 cells (Graham et        al., 1977) and 911 cells (Fallaux et al., 1996) that are        E1-transformed human embryonic kidney (“HEK”) cells and human        embryonic retinoblasts (“HER”), respectively;    -   5) After transfection of HER cells with construct pIG.E1B (see        FIGS. 4A and 4B), seven independent cell lines could be        established. These cell lines were designated PER.C1, PER.C3,        PER.C4, PER.C5, PER.C6™, PER.C8, and PER.C9. PER denotes        PGK-E1-Retinoblasts. These cell lines express E1A and E1B        proteins, are stable (e.g., PER.C6™ for more than 57 passages),        and complement E1-defective adenovirus vectors. Yields of        recombinant adenovirus obtained on PER cells are a little higher        than obtained on 293 cells. One of these cell lines (PER.C6™)        has been deposited at the ECACC™ under number 96022940;    -   6) New adenovirus vectors with extended E1 deletions (deletion        nt. 459–3510). Those viral vectors lack sequences homologous to        E1 sequences in the packaging cell lines. These adenoviral        vectors contain pIX promoter sequences and the pIX gene, as pIX        (from its natural promoter sequences) can only be expressed from        the vector and not by packaging cells (Matsui et al., 1986,        Hoeben and Fallaux, personal communication; Imler et al., 1996);    -   7) E2A-expressing packaging cell lines preferably based on        either E1A-expressing established cell lines or        E1A–E1B-expressing diploid cells (see under 2–4). Either E2A        expression is under the control of an inducible promoter or the        E2A ts125 mutant is driven by either an inducible or a        constitutive promoter.    -   8) Recombinant adenovirus vectors as described before (see 6)        but carrying an additional deletion of E2A sequences;    -   9) Adenovirus packaging cells from monkey origin that are able        to trans-complement E1-defective recombinant adenoviruses. They        are preferably co-transfected with pIG.E1A.E1B and pIG.NEO and        selected for NEO resistance. Such cells E1A and E1B are able to        transcomplement E1-defective recombinant human adenoviruses but        will do so inefficiently because of a block of the synthesis of        late adenovirus proteins in cells of monkey origin (Klessig and        Grodzicker, 1979). To overcome this problem, we generate        recombinant adenoviruses that harbor a host-range mutation in        the E2A gene, allowing human adenoviruses to replicate in monkey        cells. Such viruses are generated as described in FIG. 12,        except DNA from an hr-mutant is used for homologous        recombination; and    -   10) Adenovirus packaging cells from monkey origin as described        under 9, except that they will also be co-transfected with E2A        sequences harboring the hr mutation. This allows replication of        human adenoviruses lacking E1 and E2A (see under 8). E2A in        these cell lines is either under the control of an inducible        promoter or the tsE2A mutant is used. In the latter case, the        E2A gene will thus carry both the ts mutation and the hr        mutation (derived from ts400). Replication competent human        adenoviruses have been described that harbor both mutations        (Brough et al., 1985; Rice and Klessig, 1985).

A further aspect of the invention provides otherwise improved adenovirusvectors, as well as novel strategies for generation and application ofsuch vectors and a method for the intracellular amplification of linearDNA fragments in mammalian cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures and drawings may help to understand the invention:

FIG. 1 illustrates the construction of pBS.PGK.PCRI;

FIG. 2 illustrates the construction of pIG.E1A.E1B.X;

FIGS. 3A and 3B illustrate the construction of pIG.E1A.NEO;

FIGS. 4A and 4B illustrate the construction of pIG.E1A.E1B;

FIG. 5 illustrates the construction of pIG.NEO;

FIG. 6 illustrates the transformation of primary baby rat kidney (“BRK”)cells by adenovirus packaging constructs;

FIG. 7 illustrates a Western blot analysis of A549 clones transfectedwith pIG.E1A.NEO and HER cells transfected with pIG.E1A.E1B (PERclones);

FIG. 8 illustrates a Southern blot analysis of 293, 911 and PER celllines. Cellular DNA was extracted, HindIII digested, electrophoresed andtransferred to Hybond N+membranes (Amersham);

FIG. 9 illustrates the transfection efficiency of PER.C3, PER.C5,PER.C6™ and 911 cells;

FIG. 10 illustrates construction of adenovirus vector pMLPI.TK. pMLPI.TKis designed to have no sequence overlap with the packaging constructpIG.E1A.E1B;

FIGS. 11A and 11B illustrate new adenovirus packaging constructs whichdo not have sequence overlap with new adenovirus vectors;

FIG. 12 illustrates the generation of recombinant adenovirusIG.Ad.MLPI.TK;

FIG. 13 illustrates the adenovirus double-stranded DNA genome indicatingthe approximate locations of E1, E2, E3, E4, and L regions;

FIG. 14 illustrates the adenovirus genome shown in the top left with theorigins of replication located within the left and right ITRs at thegenome ends;

FIG. 15 illustrates a potential hairpin conformation of asingle-stranded DNA molecule that contains the HP/asp sequence (SEQ IDNO:22);

FIG. 16 illustrates a diagram of pICLhac;

FIG. 17 illustrates a diagram of pICLhaw;

FIG. 18 illustrates a schematic representation of pICLI;

FIG. 19 is a diagram of pICL (SEQ ID NO:21); and

DETAILED DESCRIPTION

The so-called “minimal” adenovirus vectors according to the presentinvention retain at least a portion of the viral genome that is requiredfor encapsidation of the genome into virus particles (the encapsidationsignal), as well as at least one copy of at least a functional part or aderivative of the ITR, that is, DNA sequences derived from the terminiof the linear adenovirus genome. The vectors according to the presentinvention will also contain a transgene linked to a promoter sequence togovern expression of the transgene. Packaging of the so-called minimaladenovirus vector can be achieved by coinfection with a helper virus or,alternatively, with a packaging-deficient replicating helper system, asdescribed below.

Adenovirus-derived DNA fragments that can replicate in suitable celllines and that may serve as a packaging-deficient replicating helpersystem are generated as follows. These DNA fragments retain at least aportion of the transcribed region of the “late” transcription unit ofthe adenovirus genome and carry deletions in at least a portion of theE1 region and deletions in at least a portion of the encapsidationsignal. In addition, these DNA fragments contain at least one copy of anITR. An ITR is located at one terminus of the transfected DNA molecule.The other end may contain an ITR or, alternatively a DNA sequence thatis complementary to a portion of the same strand of the DNA moleculeother than the ITR. If, in the latter case, the two complementarysequences anneal, the free 3′-hydroxyl group of the 3′ terminalnucleotide of the hairpin structure can serve as a primer for DNAsynthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion of the displaced strand into a double-strandedform of at least a portion of the DNA molecule. Further replicationinitiating at the ITR will result in a linear double-stranded DNAmolecule that is flanked by two ITRs and is larger than the originaltransfected DNA molecule (see FIG. 13). This molecule can replicateitself in the transfected cell by virtue of the adenovirus proteinsencoded by the DNA molecule and the adenoviral and cellular proteinsencoded by genes in the host-cell genome. This DNA molecule cannot beencapsidated due to its large size (greater than 39,000 base pairs) ordue to the absence of a functional encapsidation signal. This DNAmolecule is intended to serve as a helper for the production ofdefective adenovirus vectors in suitable cell lines.

The invention also comprises a method for amplifying linear DNAfragments of variable sizes in suitable mammalian cells. These DNAfragments contain at least one copy of the ITR at one of the termini ofthe fragment. The other end may contain an ITR or, alternatively, a DNAsequence that is complementary to a portion of the same strand of theDNA molecule other than the ITR. If, in the latter case, the twocomplementary sequences anneal, the free 3′-hydroxyl group of the 3′terminal nucleotide of the hairpin structure can serve as a primer forDNA synthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion of the displaced strand into a double-strandedform of at least a portion of the DNA molecule. Further replicationinitiating at the ITR will result in a linear double-stranded DNAmolecule that is flanked by two ITRs, which is larger than the originaltransfected DNA molecule. A DNA molecule that contains ITR sequences atboth ends can replicate itself in transfected cells by virtue of thepresence of at least the adenovirus E2 proteins (viz. the DNA-bindingprotein (“DBP”), the adenovirus DNA polymerase (“Ad-pol”), and thepreterminal protein (“pTP”). The required proteins may be expressed fromadenovirus genes on the DNA molecule itself, from adenovirus E2 genesintegrated in the host-cell genome, or from a replicating helperfragment, as described above.

Several groups have shown that the presence of ITR sequences at the endof DNA molecules is sufficient to generate adenovirus minichromosomesthat can replicate if the adenovirus-proteins required for replicationare provided in trans, for example, by infection with a helper virus (Huet al., 1992; Wang and Pearson, 1985; Hay et al., 1984). Hu et al;,(1992) observed the presence and replication of symmetrical adenovirusminichromosome-dimers after transfection of plasmids containing a singleITR. The authors were able to demonstrate that these dimericminichromosomes arise after tail-to-tail ligation of the single ITR DNAmolecules. In DNA extracted from defective adenovirus type 2 particles,dimeric molecules of various sizes have also been observed usingelectron-microscopy (Daniell, 1976). It was suggested that theincomplete genomes were formed by illegitimate recombination betweendifferent molecules and that variations in the position of the sequenceat which the illegitimate base pairing occurred were responsible for theheterogeneous nature of the incomplete genomes. Based on this mechanism,it was speculated that, in theory, defective molecules with a totallength of up to two times the normal genome could be generated. Suchmolecules could contain duplicated sequences from either end of thegenome. However, no DNA molecules larger than the full-length virus werefound packaged in the defective particles (Daniell, 1976). This can beexplained by the size limitations that apply to the packaging. Inaddition, it was observed that, in the virus particles, DNA moleculeswith a duplicated left end predominated over those containing theright-end terminus (Daniell, 1976). This is fully explained by thepresence of the encapsidation signal near that left end of the genome(Gräble and Hearing, 1990; Gräble and Hearing, 1992; Hearing et al.,1987).

The major problems associated with the current adenovirus-derivedvectors are:

-   -   1) The strong immunogenicity of the virus particle;    -   2) The expression of adenovirus genes that reside in the        adenoviral vectors, resulting in a Cytotoxic T-cell response        against the transduced cells; and    -   3) The low amount of heterologous sequences that can be        accommodated in the current vectors (up to maximally        approximately 8,000 base pairs (“bp”) of heterologous DNA).

The strong immunogenicity of the adenovirus particle results in animmunological response of the host, even after a single administrationof the adenoviral vector. As a result of the development of neutralizingantibodies, a subsequent administration of the virus will be lesseffective or even completely ineffective. However, a prolonged orpersistent expression of the transferred genes will reduce the number ofadministrations required and may bypass the problem.

With regard to problem 2), experiments performed by Wilson andcollaborators have demonstrated that after adenovirus-mediated genetransfer into immunocompetent animals, the expression of the transgenegradually decreases and disappears approximately 2–4 weekspost-infection (Yang et al., 1994a; Yang et al., 1994b). This is causedby the development of a Cytotoxic T-Cell (“CTL”) response against thetransduced cells. The CTLs were directed against adenovirus proteinsexpressed by the viral vectors. In the transduced cells, synthesis ofthe adenovirus DNA-binding protein (the E2A-gene product), penton, andfiber proteins (late-gene products) could be established. Theseadenovirus proteins, encoded by the viral vector, were expressed despitedeletion of the E1 region. This demonstrates that deletion of the E1region is not sufficient to completely prevent expression of the viralgenes (Engelhardt et al., 1994a).

With regard to problem 3), studies by Graham and collaborators havedemonstrated that adenoviruses are capable of encapsidating DNA of up to105% of the normal genome size (Bett et al., 1993). Larger genomes tendto be unstable, resulting in loss of DNA sequences during propagation ofthe virus. Combining deletions in the E1 and E3 regions of the viralgenomes increases the maximum size of the foreign DNA that can beencapsidated to approximately 8.3 kb. In addition, some sequences of theE4 region appear to be dispensable for virus growth (adding another 1.8kb to the maximum encapsidation capacity). Also, the E2A region can bedeleted from the vector when the E2A gene product is provided in transin the encapsidation cell line, adding another 1.6 kb. It is, however,unlikely that the maximum capacity of foreign DNA can be significantlyincreased further than 12 kb.

We developed a new strategy for the generation and production ofhelper-free stocks of recombinant adenovirus vectors that canaccommodate up to 38 kb of foreign DNA. Only two functional ITRsequences and sequences that can function as an encapsidation signalneed to be part of the vector genome. Such vectors are called “minimaladenovectors.” The helper functions for the minimal adenovectors areprovided in trans by encapsidation defective-replication competent DNAmolecules that contain all the viral genes encoding the required geneproducts, with the exception of those genes that are present in thehost-cell genome, or genes that reside in the vector genome.

The applications of the disclosed inventions are outlined below and willbe illustrated in the experimental part, which is only intended for thatpurpose and should not be used to reduce the scope of the presentinvention as understood by those skilled in the art.

Use of the IG Packaging Constructs Diploid Cells.

The constructs, in particular pIG.E1A.E1B, will be used to transfectdiploid human cells, such as HER, HEK, and Human Embryonic Lung cells(“HEL”). Transfected cells will be selected for transformed phenotype(focus formation) and tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such celllines will be used for the generation and (large-scale) production ofE1-deleted recombinant adenoviruses. Such cells infected withrecombinant adenovirus, are also intended to be used in vivo as a localproducer of recombinant adenovirus, for example, for the treatment ofsolid tumors.

911 cells are used for the titration, generation, and production ofrecombinant adenovirus vectors (Fallaux et al., 1996).

HER cells transfected with pIG.E1A.E1B have resulted in 7 independentclones (called PER cells). These clones are used for the production ofE1-deleted (including non-overlapping adenovirus vectors) orE1-defective recombinant adenovirus vectors and provide the basis forintroduction of, for example, E2B or E2A constructs (e.g., ts125E2A, seebelow), E4, etc., that will allow propagation of adenovirus vectors thathave mutations in, for example, E2A or E4.

In addition, diploid cells of other species that are permissive forhuman adenovirus, such as the cotton rat (Sigmodon hispidus) (Pacini etal., 1984), Syrian hamster (Morin et al., 1987), or chimpanzee (Levreroet al., 1991), will be immortalized with these constructs. Such cellsinfected with recombinant adenovirus are also intended to be used invivo for the local production of recombinant adenovirus, for example,for the treatment of solid tumors.

Established Cells.

The constructs, in particular pIG.E1A.NEO, can be used to transfectestablished cells, for example, A549 (human bronchial carcinoma), KB(oral carcinoma), MRC-5 (human diploid lung cell line), or GLC celllines (small cell lung cancer) (de Leij et al., 1985; Postmus et al.,1988) and selected for NEO resistance. Individual colonies of resistantcells are isolated and tested for their capacity to support propagationof E1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Whenpropagation of E1-deleted viruses on E1A-containing cells is possible,such cells can be used for the generation and production of E1-deletedrecombinant adenovirus. They can also be used for the propagation ofE1A-deleted/E1B-retained recombinant adenovirus.

Established cells can also be co-transfected with pIG.E1A.E1B andpIG.NEO (or another NEO-containing expression vector). Clones resistantto G418 are tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK, and used forthe generation and production of E1-deleted recombinant adenovirus andwill be applied in vivo for local production of recombinant virus, asdescribed for the diploid cells (see previous discussion). All celllines, including transformed diploid cell lines or NEO-resistantestablished lines, can be used as the basis for the generation of “nextgeneration” packaging cell lines that support propagation ofE1-defective recombinant adenoviruses and that also carry deletions inother genes, such as E2A and E4. Moreover, they will provide the basisfor the generation of minimal adenovirus vectors as disclosed herein.

E2-Expressing Cell Lines.

Packaging cells expressing E2A sequences are and will be used for thegeneration and large-scale production of E2A-deleted recombinantadenovirus.

The newly generated human adenovirus packaging cell lines or cell linesderived from species permissive for human adenovirus (E2A or ts125E2A;E1A+E2A; E1A+E1B+E2A; E1A−E2A/ts125; E1A+E1B−E2A/ts125) or nonpermissivecell lines such as monkey cells (hrE2A or hr+ts125E2A; E1A+hrE2A;E1A+E1B+hrE2A; E1A+hrE2A/ts125; E1A−E1B+hrE2A/ts125) are and will beused for the generation and large-scale production of E2A-deletedrecombinant adenovirus vectors. In addition, they will be applied invivo for local production of recombinant virus, as described for thediploid cells (see previous discussion).

Novel Adenovirus Vectors.

The newly developed adenovirus vectors harboring an E1 deletion of nt.459–3510 will be used for gene transfer purposes. These vectors are alsothe basis for the development of further deleted adenovirus vectors thatare mutated for, for example, E2A, E2B or E4. Such vectors will begenerated, for example, on the newly developed packaging cell linesdescribed above.

Minimal Adenovirus Packaging System.

We disclose adenovirus packaging constructs (to be used for thepackaging of minimal adenovirus vectors) which have the followingcharacteristics:

-   -   1) the packaging construct replicates;    -   2) the packaging construct cannot be packaged because the        packaging signal is deleted;    -   3) the packaging construct contains an internal hairpin-forming        sequence (see FIG. 15);    -   4) because of the internal hairpin structure, the packaging        construct is duplicated, that is, the DNA of the packaging        construct becomes twice as long as it was before transfection        into the packaging cell (in our sample, it duplicates from 35 kb        to 70 kb). This duplication also prevents packaging. Note that        this duplicated DNA molecule has ITRs at both termini (see,        e.g., FIG. 13);    -   5) this duplicated packaging molecule is able to replicate like        a “normal adenovirus” DNA molecule;    -   6) the duplication of the genome is a prerequisite for the        production of sufficient levels of adenovirus proteins required        to package the minimal adenovirus vector; and    -   7) the packaging construct has no overlapping sequences with the        minimal vector or cellular sequences that may lead to generation        of RCA by homologous recombination.

This packaging system is used to produce minimal adenovirus vectors. Theadvantages of minimal adenovirus vectors, for example, for gene therapyof vaccination purposes, are well known (accommodation of up to 38 kb;gutting of potentially toxic and immunogenic adenovirus genes).

Adenovirus vectors containing mutations in essential genes (includingminimal adenovirus vectors) can also be propagated using this system.

Use of Intracellular E2-Expressing Vectors.

Minimal adenovirus vectors are generated using the helper functionsprovided in trans by packaging-deficient replicating helper molecules.The adenovirus-derived ITR sequences serve as origins of DNA replicationin the presence of at least the E2-gene products. When the E2-geneproducts are expressed from genes in the vector genome (the gene(s) mustbe driven by an E1-independent promoter), the vector genome canreplicate in the target cells. This will allow a significantly increasednumber of template molecules in the target cells and, as a result, anincreased expression of the genes of interest encoded by the vector.This is of particular interest for application of gene therapy in cancertreatment.

Applications of Intracellular Amplification of Linear DNA Fragments.

A similar approach could also be taken if amplification of linear DNAfragments is desired. DNA fragments of known or unknown sequence couldbe amplified in cells containing the E2 gene products if at least oneITR sequence is located near or at its terminus. There are no apparentconstraints on the size of the fragment. Even fragments much larger thanthe adenovirus genome (36 kb) should be amplified using this approach.It is thus possible to clone large fragments in mammalian cells withouteither shuttling the fragment into bacteria (such as E. coli) or usingthe polymerase chain reaction (“PCR”). At the end stage of a productiveadenovirus infection, a single cell can contain over 100,000 copies ofthe viral genome. In the optimal situation, the linear DNA fragments canbe amplified to similar levels. Thus, one should be able to extract morethan 5 μg of DNA fragment per 10 million cells (for a 35-kbp fragment).This system can be used to express heterologous proteins equivalent tothe Simian Virus 40-based COS cell system for research or fortherapeutic purposes. In addition, the system can be used to identifygenes in large fragments of DNA. Random DNA fragments may be amplified(after addition of ITRs) and expressed during intracellularamplification. Election or selection of those cells with the desiredphenotype can be used to enrich the fragment of interest and to isolatethe gene.

Experiments

Generation of Cell Lines Able to Transcomplement E1-DefectiveRecombinant Adenovirus Vectors.

-   -   911 cell line

We have generated a cell line that harbors E1 sequences of adenovirustype 5 (“Ad5”) and is able to trans-complement E1-deleted recombinantadenovirus (Fallaux et al., 1996). This cell line was obtained bytransfection of diploid human embryonic retinoblasts (“HER”) withpAd5XhoIC which contains nt. 80–5788 of Ad5. One of the resultingtransformants was designated 911. This cell line has been shown to bevery useful in the propagation of E1-defective recombinant adenovirus.It was found to be superior to 293 cells. Unlike 293 cells, 911 cellslack a fully transformed phenotype, which most likely is the cause ofits better performance as an adenovirus packaging line:

-   -   1) plaque assays can be performed faster (4–5 days instead of        8–14 days, as on 293),    -   2) monolayers of 911 cells survive better under agar overlay, as        required for plaque assays, and    -   3) higher amplification of E1-deleted vectors is obtained.

In addition, unlike 293 cells that were transfected with shearedadenoviral DNA, 911 cells were transfected using a defined construct.Transfection efficiencies of 911 cells are comparable to those of 293.

New Packaging Constructs. Source of Adenovirus Sequences.

Adenovirus sequences are derived either from pAd5.SalB containing nt.80–9460 of human adenovirus type 5 (Bernards et al., 1983), or fromwild-type Ad5 DNA. pAd5.SalB was digested with SalI and XhoI, the largefragment was relegated, and this new clone was named pAd5.X/S. The pTNconstruct (constructed by Dr. R. Vogels, IntroGene, Leiden, TheNetherlands) was used as a source for the human PGK promoter and the NEOgene.

Human PGK Promoter and NEO^(R) Gene.

Transcription of E1A sequences in the new packaging constructs is drivenby the human PGK promoter (Michelson et al., 1983; Singer-Sam et al.,1984), derived from plasmid pTN (gift of R. Vogels), which uses pUC119(Vieira and Messing, 1987) as a backbone. This plasmid was also used asa source for the NEO gene fused to the Hepatitis B Virus (“HBV”)poly-adenylation signal.

Fusion of PGK Promoter to E1 Genes.

As shown in FIG. 1, in order to replace the E1 sequences of Ad5 (ITR,origin of replication, and packaging signal) by heterologous sequences,we have amplified E1 sequences (nt. 459 to nt. 960) of Ad5 by PCR, usingprimers Ea-1 (SEQ ID NO:1) and Ea-2 (SEQ ID NO:2) (see Table I). Theresulting PCR product was digested with ClaI and ligated into Bluescript(Stratagene), predigested with ClaI and EcoRV, resulting in constructpBS.PCR1.

TABLE I Name (SEQ ID NO) Sequence Function Primer Ea-1 CGTGTAGTGTATTTATACCC PCR amplification Ad5 nt459 -> (SEQ ID NO: 1) a G Primer Ea-2TCGTCACTGG GTGGAAAGCC PCR amplification Ad5 nt960 <- (SEQ ID NO: 2) a APrimer Ea-3 TACCCGCCGT CCTAAAATGG nt1284–1304 of Ad5 genome (SEQ ID NO:3) a C Primer Ea-5 TGGACTTGAG CTGTAAACGC nt1514–1533 of Ad5 genome (SEQID NO: 4) a Primer Ep-2 GCCTCCATGG AGGTCAGATG nt1721–1702 of Ad5: (SEQID NO: 5) a T introduction of NcoI site Primer Eb-1 GCTTGAGCCCGAGACATGTC nt3269–3289 of Ad5 genome (SEQ ID NO: 6) a Primer Eb-2CCCCTCGAGC TCAATCTGTA nt3508–3496 of Ad5 genome: (SEQ ID NO: 7) a TCTTintroduction of XhoI site Primer SV40-1 GGGGGATCCG AACTTGTTTAIntroduction BamHI site (nt2182– (SEQ ID NO: 8) a TTGCAGC 2199 ofpMLP.TK) adaptation of recombinant adenoviruses Primer SV40-2 GGGAGATCTAGACATGATAA Introduction BglII site (nt2312–2297 (SEQ ID NO: 9) a GATACof pMLP.TK) Primer Ad5-1 GGGAGATCTG TACTGAAATG Introduction of BglIIsite (nt 2496– (SEQ ID NO: 10) a TGTGGGC 2514 of pMLP.TK) Primer Ad5-2GGAGGCTGCA GTCTCCAACG Rnt2779–2756 of PMLP.TK (SEQ ID NO: 11) a GCGTPrimer ITR1 GGGGGATCCT CAAATCGTCA nt35737–35757 of Ad5 (introduction(SEQ ID NO: 12) a CTTCCGT of BamHI site) Primer ITR2 GGGGTCTAGACATCATCAAT nt35935–35919 of Ad5 (introduction (SEQ ID NO: 13) a AATATACof XbaI site) PCR primer PCR/MLP1 GGCGAATTCG TCGACATCAT (Ad5 nt. 10–18)(SEQ ID NO: 14) b CAATAATATA CC PCT primer PCR/MLP2 GGCGAATTCGGTACCATCAT (Ad5 nt. 10–18) (SEQ ID NO: 15) b CAATAATATA CC PCT primerPCR/MLP3 CTGTGTACAC CGGCGCA (Ad5 nt. 200–184) (SEQ ID NO: 16) b PCTprimer HP/asp1 5′-GTACACTGAC CTAGTGCCGC (SEQ ID NO: 17) c CCGGGCAAAGCCCGGGCGGC ACTAGGTCAG PCT primer HP/asp2 5′-GTACCTGACC TAGTGCCGCC (SEQID NO: 18) c CGGGCTTTGC CCGGGCGGCA CTAGGTCAGT PCT primer HP/cla15′-GTACATTGAC CTAGTGCCGC (SEQ ID NO: 19) d CCGGGCAAAG CCCGGGCGGCACTAGGTCAA TCGAT PCT primer HP/cla2 5′-GTACATCGAT TGACCTAGTG (SEQ ID NO:20) d CCGCCCGGGC TTTGCCCGGG CGGCACTAGG TCAAT Table I. Primer Sequences.a Primers used for PCR amplification of DNA fragments used forgeneration of constructs described in this patent application. b PCRprimer sets to be used to create the SalI and Asp718 sites juxtaposed tothe ITR sequences. c Synthetic oligonucleotide pair used to generate asynthetic hairpin, recreates an Asp718 site at one of the termini ifinserted in the Asp718 site. d Synthetic oligonucleotide pair used togenerate a synthetic hairpin, contains the ClaI recognition site to beused for hairpin formation.

Vector pTN was digested with restriction enzymes EcoRI (partially) andScaI and the DNA fragment containing the PGK promoter sequences wasligated into PBS.PCRI digested with ScaI and EcoRI. The resultingconstruct pBS.PGK.PCR1 contains the human PGK promoter operativelylinked to Ad5 E1 sequences from nt. 459 to nt. 916.

Construction of pIG.E1A.E1B.X.

As shown in FIG. 2, pIG.E1A.E1B.X was made by replacing the ScaI-BspE1fragment of pAT.X/S by the corresponding fragment from pBS.PGK.PCR1(containing the PGK promoter linked to E1A sequences). pIG.E1A.E1B.Xcontains the E1A and E1B coding sequences under the direction of the PGKpromoter. As Ad5 sequences from nt. 459 to nt. 5788 are present in thisconstruct, pIX protein of adenovirus is also encoded by this plasmid.

Construction of pIG.E1A.NEO.

As shown in FIG. 3A, in order to introduce the complete E1B promoter andto fuse this promoter in such a way that the AUG codon of E1B 21 kDafunctions exactly as the AUG codon of NEO^(R), we amplified the E1Bpromoter using primers Ea-3 (SEQ ID NO:3) and Ep-2 (SEQ ID NO:5), whereprimer Ep-2 (SEQ ID NO:5) introduces an NcoI site in the PCR fragment.The resulting PCR fragment, named PCRII, was digested with HpaI and NcoIand ligated into pAT-X/S, which was predigested with HpaI and with NcoI.The resulting plasmid was designated pAT.X/S.PCR2. The NcoI-StuIfragment of pTN, containing the NEO gene and part of the HBVpolyadenylation signal, was cloned into pAT.X/S.PCR2 (digested with NcoIand NruI). The resulting construct was pAT.PCR2.NEO.

As shown in FIG. 3B, the polyadenylation signal was completed byreplacing the ScaI-SalI fragment of pAT-PCR2-NEO with the correspondingfragment of pTN (resulting in pAT.PCR2.NEO.p(A)). The ScaI-XbaI ofpAT.PCR2.NEO.p (A) was replaced by the corresponding fragment of pIG.E1A.E1B.X, containing the PGK promoter linked to E1A genes. The resultingconstruct was named pIG.E1A.NEO, and thus contains Ad5 E1 sequences (nt.459 to nt. 1713) under the control of the human PGK promoter.

Construction of pIG.E1A.E1B.

As shown in FIGS. 4A and 4B, pIG.E1A.E1B was made by amplifying thesequences encoding the N-terminal amino acids of E1B 55 kDa usingprimers Eb-1 (SEQ ID NO:6) and Eb-2 (SEQ ID NO:7) (introduces an XhoIsite). The resulting PCR fragment was digested with BglII and clonedinto BglII/NruI of pAT-X/S, thereby obtaining pAT.PCR3.

pIG.E1A.E1B was constructed by introducing the HBV poly(A) sequences ofpIG.E1A.NEO downstream of E1B sequences of pAT.PCR3 by exchange of theXbaI-SalI fragment of pIG.E1A.NEO and the XbaI-XhoI fragment ofpAT.PCR3.

pIG.E1A.E1B contains nt. 459 to nt. 3510 of Ad5, which encode the E1Aand E1B proteins. The E1B sequences are terminated at the spliceacceptor at nt. 3511. No pIX sequences are present in this construct.

Construction of pIG.NEO.

As shown in FIG. 5, pIG.NEO was generated by cloning the HpaI-ScaIfragment of pIG.E1A.NEO, containing the NEO gene under the control ofthe Ad.5 E1B promoter, into pBS digested with EcoRV and ScaI.

This construct is of use when established cells are transfected withE1A.E1B constructs and NEO selection is required. Because NEO expressionis directed by the E1B promoter, NEO resistant cells are expected toco-express E1A, which is also advantageous for maintaining high levelsof expression of E1A during long-term culture of the cells.

Testing of Constructs.

The integrity of the constructs pIG.E1A.NEO, pIG.E1A.E1B.X andpIG.E1A.E1B was assessed by restriction enzyme mapping; furthermore,parts of the constructs that were obtained by PCR analysis wereconfirmed by sequence analysis. No changes in the nucleotide sequencewere found.

The constructs were transfected into primary Baby Rat Kidney (“BRK”)cells and tested for their ability to immortalize (pIG.E1A.NEC) or fullytransform (pAd5.XhoIC, pIG.E1A.E1B.X, and pIG.E1A.E1B) these cells.

Kidneys of 6-day old WAG-Rij rats were isolated, homogenized, andtrypsinized. Subconfluent dishes (diameter 5 cm) of the BRK cellcultures were transfected with 1 or 5 μg of pIG.NEO, pIG.E1A.NEO,pIG.E1A.E1B, pIG.E1A.E1B.X, pAd5.XhoIC, or pIG.E1A.NEO together withPDC26 (Van der E1sen et al., 1983), carrying the Ad5.E1B gene undercontrol of the SV40 early promoter. Three weeks post-transfection, whenfoci were visible, the dishes were fixed, Giemsa stained, and the focicounted.

An overview of the generated adenovirus packaging constructs and theirability to transform BRK is presented in FIG. 6. The results indicatethat the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transformBRK cells in a dose-dependent manner. The efficiency of transformationis similar for both constructs and is comparable to what was found withthe construct that was used to make 911 cells, namely pAd5.XhoIC.

As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However,co-transfection of an E1B expression construct (PDC26) did result in asignificant increase in the number of transformants (18 versus 1),indicating that E1A encoded by pIG.E1A.NEO is functional. We conclude,therefore, that the newly generated packaging constructs are suited forthe generation of new adenovirus packaging lines.

Generation of Cell Lines with New packaging Constructs, Cell Lines, andCell Culture.

Human A549 bronchial carcinoma cells (Shapiro et al., 1978), humanembryonic retinoblasts (“HER”), Ad5-E1-transformed human embryonickidney (“HEK”) cells (293; Graham et al., 1977), and Ad5-transformed HERcells (911; Fallaux et al., 1996) and PER cells were grown in Dulbecco'sModified Eagle Medium (“DMEM”) supplemented with 10% Fetal Calf Serum(“FCS”) and antibiotics in a 5% CO₂ atmosphere at 37° C. Cell culturemedia, reagents, and sera were purchased from Gibco Laboratories (GrandIsland, N.Y.). Culture plastics were purchased from Greiner (Nürtingen,Germany) and Corning (Corning, N.Y.).

Viruses and Virus Techniques.

The construction of adenoviral vectors IG.Ad.MLP.nls.lacZ,IG.Ad.MLP.luc, IG.Ad.MLP.TK, and IG.Ad.CMV.TK is described in detail inEuropean patent application EP 95202213. The recombinant adenoviralvector IG.Ad.MLP.nls.lacZ contains the E. coli lacZ gene, encodingβZgalactosidase, under control of the Ad2 major late promoter (“MLP”).IG.Ad.MLP.luc contains the firefly luciferase gene driven by the Ad2MLP. Adenoviral vectors IG.Ad.MLP.TK and IG.Ad.CMV.TR contain the HerpesSimplex Virus thymidine kinase (“TK”) gene under the control of the Ad2MLP and the Cytomegalovirus (“CMV”) enhancer/promoter, respectively.

Transfections.

All transfections were performed by calcium-phosphate precipitation DNA(Graham and Van der Eb, 1973) with the GIBCO Calcium PhosphateTransfection System (GIBCO BRL Life Technologies Inc., Gaithersburg,Md., USA), according to the manufacturer's protocol.

Western Blotting.

Subconfluent cultures of exponentially growing 293, 911 andAd5-E1-transformed A549 and PER cells were washed with PBS and scrapedin Fos-RIPA buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP4O, 1% sodiumdodecyl sulphate (“SDS”), 1% NA-DOC, 0.5 mM phenyl methyl sulphonylfluoride (“PMSF”), 0.5 mM trypsin inhibitor, 50 mM NaF and 1 mM sodiumvanadate). After 10 minutes at room temperature, lysates were cleared bycentrifugation. Protein concentrations were measured with the Bioradprotein assay kit, and 25 μg total cellular protein was loaded on a12.5% SDS-PAA gel. After electrophoresis, proteins were transferred tonitrocellulose (1 hour at 300 mA). Prestained standards (Sigma, USA)were run in parallel. Filters were blocked with 1% bovine serum albumin(“BSA”) in TBST (10 mM Tris, pH 8.15 mM NaCl, and 0.05% TWEEN™-20) for 1hour. The first antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDaantibody A1C6 (Zantema et al., unpublished) and the rat monoclonalanti-Ad5-E1B-221-kDa antibody C1G11 (Zantema et al., 1985). The secondantibody was a horseradish peroxidase-labeled goat anti-mouse antibody(Promega). Signals were visualized by enhanced chemiluminescence(Amersham Corp, UK).

Southern Blot Analysis.

High molecular weight DNA was isolated and 10 μg was digested tocompletion and fractionated on a 0.7% agarose gel. Southern blottransfer to Hybond N+ (Amersham, UK) was performed with a 0.4 M NaOH,0.6 M NaCl transfer solution (Church and Gilbert, 1984). Hybridizationwas performed with a 2463-nt SspI-HindIII fragment from pAd5.SalB(Bernards et al., 1983). This fragment consists of Ad5 bp. 342–2805. Thefragment was radiolabeled with αZ^(32p)-dCTP with the use of randomhexanucleotide primers and Klenow DNA polymerase. The Southern blotswere exposed to a Kodak XAR-5 film at −80° C. and to a Phospho-Imagerscreen that was analyzed by B&L Systems' Molecular Dynamics software.

A549.

Ad5-E1-transformed A549 human bronchial carcinoma cell lines weregenerated by transfection with pIG.E1A.NEO and selection forG418resistance. Thirty-one G418 resistant clones were established.Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418 resistantcell lines.

PER.

Ad5-E1-transformed HER cells were generated by transfection of primaryHER cells with plasmid pIG.E1A.E1B. Transformed cell lines wereestablished from well-separated foci. We were able to establish sevenclonal cell lines, which we called PER.C1, PER.C3, PER.C4, PER.C5,PER.C6™, PER.C8, and PER.C9. One of the PER clones, namely PER.C6™, hasbeen deposited under the Budapest Treaty under number ECACC™ 96022940with the Centre for Applied Microbiology and Research of Porton Down,UK, on Feb. 29, 1996. In addition, PER.C6™ is commercially availablefrom IntroGene, B.V., Leiden, NL.

Expression of Ad5 E1A and E1B Genes in Transformed A549 and PER Cells.

Expression of the Ad5 E1A and the 55 kDa and 21 kDa E1B proteins in theestablished A549 and PER cells was studied by means of Western blottingwith the use of monoclonal antibodies (“Mab”). Mab M73 recognizes theE1A products, whereas Mabs AIC6 and C1G11 are directed against the 55kDa and 21 kDa E1B proteins, respectively.

The antibodies did not recognize proteins in extracts from the parentalA549 or the primary HER cells (data not shown). None of the A549 clonesthat were generated by co-transfection of pIG.NEO and pIG.E1A.E1Bexpressed detectable levels of E1A or E1B proteins (not shown). Some ofthe A549 clones that were generated by transfection with pIG.E1A.NEOexpressed the Ad5 E1A proteins (see FIG. 7), but the levels were muchlower than those detected in protein lysates from 293 cells. Thesteady-state E1A levels detected in protein extracts from PER cells weremuch higher than those detected in extracts from A549-derived cells. AllPER cell lines expressed similar levels of E1A proteins (FIG. 7). Theexpression of the E1B proteins, particularly in the case of E1B 55 kDa,was more variable. Compared to 911 and 293, the majority of the PERclones express high levels of E1B 55 kDa and 21 kDa. The steady-statelevel of E1B 21 kDa was the highest in PER.C3. None of the PER cloneslost expression of the Ad5 E1 genes upon serial passage of the cells(not shown). We found that the level of E1 expression in PER cellsremained stable for at least 100 population doublings. We decided tocharacterize the PER clones in more detail.

Southern Analysis of PER Clones.

To study the arrangement of the Ad5-E1 encoding sequences in the PERclones, we performed Southern analyses. Cellular DNA was extracted fromall PER clones and from 293 and 911 cells. The DNA was digested withHindIII, which cuts once in the Ad5 E1region. Southern hybridization onHindIII-digested DNA, using a radiolabeled Ad5-E1-specific probe,revealed the presence of several integrated copies of pIG.E1A.E1B in thegenome of the PER clones. FIG. 8 shows the distribution pattern of E1sequences in the high molecular weight DNA of the different PER celllines. The copies are concentrated in a single band, which suggests thatthey are integrated as tandem repeats. In the case of PER.C3, C5, C6,and C9, we found additional hybridizing bands of low molecular weightthat indicate the presence of truncated copies of pIG.E1A.E1B. Thenumber of copies was determined with the use of a Phospho-Imager. Weestimated that PER.C1, C3, C4, C5, C6, C8, and C9 contain 2, 88, 5, 4,5, 5, and 3 copies of the Ad5 E1 coding region, respectively, and that911 and 293 cells contain 1 and 4 copies of the Ad5 E1 sequences,respectively.

Transfection Efficiency.

Recombinant adenovectors are generated by co-transfection of adaptorplasmids and the large ClaI fragment of Ad5 into 293 cells (see Europeanpatent application EP 95202213). The recombinant virus DNA is formed byhomologous recombination between the homologous viral sequences that arepresent in the plasmid and the adenovirus DNA. The efficacy of thismethod, as well as that of alternative strategies, is highly dependenton the transfectability of the helper cells. Therefore, we compared thetransfection efficiencies of some of the PER clones with 911 cells,using the E. coli βZgalactosidase-encoding lacZ gene as a reporter (seeFIG. 9).

Production of Recombinant Adenovirus.

Yields of recombinant adenovirus obtained after inoculation of 293, 911,PER.C3, PER.C5, and PER.C6™ with different adenovirus vectors arepresented in Table II. The results indicate that the yields obtained onPER cells are at least as high as those obtained on the existing celllines. In addition, the yields of the novel adenovirus vectorIG.Ad.MLPI.TK are similar or higher than the yields obtained for theother viral vectors on all cell lines tested.

TABLE II Passage IG.Ad. IG.Ad. Producer Cell number IG.Ad. CMV.lacZCMV.TK MLPI.TK d1313 Mean 293 6.0 5.8 24 34 17.5 911 8 14 34 180 59.5PER.C3 17 8 11 44 40 25.8 PER.C5 15 6 17 36 200 64.7 PER.C6 ™ 36 10 2258 320 102 Yields × 10⁻⁸ pfu/T175 flask.

Table II. Yields of different recombinant adenoviruses obtained afterinoculation of adenovirus E1 packaging cell lines 293, 911, PER.C3,PER.C5, and PER.C6™. The yields are the mean of two differentexperiments. IG.Ad.CMV.lacZ and IG.Ad.CMV.TK are described in Europeanpatent application EP 95202213. The construction of IG.Ad.MLPI.TK isdescribed in this patent application. Yields of virus per T80 flask weredetermined by plaque assay on 911 cells, as described (Fallaux, 1996#1493).

Generation of New Adenovirus Vectors.

The used recombinant adenovirus vectors (see European patent applicationEP 95202213) are deleted for E1 sequences from nt. 459 to nt. 3328.

As construct pE1A.E1B contains Ad5 sequences nucleotide(s) 459 tonucleotide(s) 3510, there is a sequence overlap of 183 nucleotide(s)between E1B sequences in the packaging construct pIG.E1A.E1B andrecombinant adenoviruses, such as, for example, IG.Ad.MLP.TK. Theoverlapping sequences were deleted from the new adenovirus vectors. Inaddition, noncoding sequences derived from lacZ, which are present inthe original constructs, were deleted as well. This was achieved (seeFIG. 10) by PCR amplification of the SV40 poly(A) sequences from pMLP.TKusing primers SV40-1 (SEQ ID NO:8) (introduces a BamHI site) and SV40-2(SEQ ID NO:9) (introduces a BglII site). In addition, Ad5 sequencespresent in this construct were amplified from nucleotide(s) 2496 (Ad5-1(SEQ ID NO:10), introduces a BglII site) to nucleotide(s) 2779 (Ad5-2(SEQ ID NO:11)). Both PCR fragments were digested with BglII and wereligated. The ligation product was PCR amplified using primers SV40-1(SEQ ID NO:8) and Ad5-2 (SEQ ID NO:11). The PCR product obtained was cutwith BamHI and AflII and was ligated into pMLP.TK predigested with thesame enzymes. The resulting construct, named pMLPI.TK, contains adeletion in adenovirus E1 sequences from nucleotide(s) 459 tonucleotide(s) 3510.

Packaging System.

The combination of the new packaging construct pIG.E1A.E1B and therecombinant adenovirus pMLPI.TK, which do not have any sequence overlap,are presented in FIGS. 11A and 11B. In these figures, the originalsituation is also presented with the sequence overlap indicated.

The absence of overlapping sequences between pIG.E1A.E1B and pMLPI.TK(FIG. 11A) excludes the possibility of homologous recombination betweenpackaging construct and recombinant virus, and is, therefore, asignificant improvement for production of recombinant adenovirus ascompared to the original situation.

In FIG. 11B, the situation is depicted for pIG.E1A.NEO andIG.Ad.MLPI.TK. pIG.E1A.NEO, when transfected into established cells, isexpected to be sufficient to support propagation of E1-deletedrecombinant adenovirus. This combination does not have any sequenceoverlap, preventing generation of RCA by homologous recombination. Inaddition, this convenient packaging system allows the propagation ofrecombinant adenoviruses that are deleted just for E1A sequences and notfor E1B sequences. Recombinant adenoviruses expressing E1B in theabsence of E1A are attractive, as the E1B protein, in particular E1B 19kDa, is able to prevent infected human cells from lysis by TumorNecrosis Factor (“TNF”) (Gooding et al., 1991).

Generation of Recombinant Adenovirus Derived from pMLPI.TK.

Recombinant adenovirus was generated by co-transfection of 293 cellswith SalI-linearized pMLPI.TK DNA and ClaI linearized Ad5 wt DNA. Theprocedure is schematically represented in FIG. 12.

Outline of the Strategy to Generate Packaging Systems for MinimalAdenovirus Vector.

Name convention of the plasmids used:

p plasmid I ITR (Adenovirus Inverted Terminal Repeat) C CMVEnhancer/Promoter Combination L Firefly Luciferase Coding Sequence hac,haw Potential hairpin that can be formed after digestion withrestriction endonuclease Asp718 in its correct orientation and in thereverse orientation, respectively (FIG. 15 (SEQ ID NO:22)).

For example, pICLhaw is a plasmid that contains the adenovirus ITRfollowed by the CMV-driven luciferase gene and the Asp718 hairpin in thereverse (nonfunctional) orientation.

Experiment Series 1.

The following demonstrates the competence of a synthetic DNA sequencethat is capable of forming a hairpin structure to serve as a primer forreverse strand synthesis for the generation of double-stranded DNAmolecules in cells that contain and express adenovirus genes.

Plasmids pICLhac, pICLhaw, pICLI and pICL (SEQ ID NO:21) were generatedusing standard techniques. The schematic representation of theseplasmids is shown in FIGS. 16–19.

Plasmid pICL (SEQ ID NO:21) is derived from the following plasmids:

nt. 1–457 pMLP10 (Levrero et al., 1991); nt. 458–1218 pCMVβ (Clontech,EMBL Bank No. U02451); nt. 1219–3016 pMLP.luc (IntroGene, Leiden, NL,unpublished); and nt. 3017–5620 pBLCAT5 (Stein and Whelan, 1989).

The plasmid has been constructed as follows:

The tet gene of plasmid pMLP10 has been inactivated by deletion of theBamHI-SalI fragment to generate pMLP10ΔSB. Using primer set PCR/MLP1(SEQ ID NO:14) and PCR/MLP3 (SEQ ID NO:16), a 210 bp fragment containingthe Ad5-ITR, flanked by a synthetic SalI restriction site, was amplifiedusing pMLP10 DNA as the template. The PCR product was digested with theenzymes EcoRI and SgrAI to generate a 196 bp fragment. Plasmid pMLP10ΔSBwas digested with EcoRI and SgrAI to remove the ITR. This fragment wasreplaced by the EcoRI-SgrAI-treated PCR fragment to generate pMLP/SAL.Plasmid pCMV-Luc was digested with PvuII to completion and recirculatedto remove the SV40-derived polyadenylation signal and Ad5 sequences withthe exception of the Ad5 left terminus. In the resulting plasmid,pCMV-lucΔAd, the Ad5 ITR was replaced by the Sal-site-flanked ITR fromplasmid pMLP/SAL by exchanging the XmnI-SacII fragments. The resultingplasmid, pCMV-lucΔAd/SAL, the Ad5 left terminus, and the CMV-drivenluciferase gene were isolated as an SalI-SmaI fragment and inserted inthe SalI- and HpaI-digested plasmid pBLCATS to form plasmid pICL (SEQ IDNO:21). Plasmid pICL is represented in FIG. 19; its sequence ispresented in SEQ ID NO:21.

The plasmid pICL (SEQ ID NO:21) contains the following features:

nt. 1–457 Ad5 left terminus (Sequence 1–457 of human adenovirus type 5);nt. 458–969 Human cytomegalovirus enhancer and immediate early promoter(see Boshart et al., A Very Strong Enhancer is Located Upstream of anImmediate Early Gene of Human Cytomegalovirus”, Cell 41, pp. 521–530(1985), hereby incorporated herein by reference) (from plasmid pCMVβ,Clontech, Palo Alto, USA); nt. 970–1204 SV40 19S exon and truncated16/19S intron (from plasmid pCMVβ); nt. 1218–2987 Firefly luciferasegene (from pMLP.luc); nt. 3018–3131 V40 tandem polyadenylation signalsfrom late transcript, derived from plasmid pBLCAT5); t. 3132–5620 UC12backbone (derived from plasmid pBLCAT5); and t. 4337–5191 βZlactamasegene (Amp-resistance gene, reverse orientation).Plasmids pICLhac and pICLhaw.

Plasmids pICLhac and pICLhaw were derived from plasmid pICL (SEQ IDNO:21) by digestion of the latter plasmid with the restriction enzymeAsp718. The linearized plasmid was treated with Calf-Intestine AlkalinePhosphatase to remove the 51 phosphate groups. The partiallycomplementary synthetic single-stranded oligonucleotide Hp/asp1 (SEQ IDNO:17) and Hp/asp2 (SEQ ID NO:18) were annealed and phosphorylated ontheir 5′ ends using T4-polynucleotide kinase.

The phosporylated double-stranded oligomers were mixed with thedephosporylated pICL fragment and ligated. Clones containing a singlecopy of the synthetic oligonucleotide inserted into the plasmid wereisolated and characterized using restriction enzyme digests. Insertionof the oligonucleotide into the Asp718 site will at one junctionrecreate an Asp718 recognition site, whereas at the other junction, therecognition site will be disrupted. The orientation and the integrity ofthe inserted oligonucleotide were verified in selected clones bysequence analyses. A clone containing the oligonucleotide in the correctorientation (the Asp718 site close to the 3205 EcoRI site) was denotedpICLhac. A clone with the oligonucleotide in the reverse orientation(the Asp718 site close to the SV40-derived poly signal) was designatedpICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and17.

Plasmid pICLI was created from plasmid pICL (SEQ ID NO:21) by insertionof the SalI-SgrAI fragment from pICL containing the Ad5-ITR into theAsp718 site of pICL. The 194 bp SalI-SgrAI fragment was isolated frompICL (SEQ ID NO:21), and the cohesive ends were converted to blunt endsusing E. coli DNA polymerase I (Klenow fragment) and dNTPs. The Asp718cohesive ends were converted to blunt ends by treatment with mung beannuclease. Clones that contain the ITR in the Asp718 site of plasmid pICL(SEQ ID NO:21) were generated by ligation. A clone that contained theITR fragment in the correct orientation was designated pICLI (see FIG.18). Generation of adenovirus Ad-CMV-hcTK recombinant adenovirus wasconstructed according to the method described in European patentapplication EP 95202213. Two components are required to generate arecombinant adenovirus. First, an adaptor-plasmid containing the leftterminus of the adenovirus genome containing the ITR and the packagingsignal, an expression cassette with the gene of interest, and a portionof the adenovirus genome which can be used for homologous recombinationare required. In addition, adenovirus DNA is needed for recombinationwith the aforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, theplasmid PCMV.TK was used as a basis. This plasmid contains nt. 1–455 ofthe adenovirus type 5 genome, nt. 456–1204 derived from pCMVβ (Clontech,the PstI-StuI fragment that contains the CMV enhancer promoter and the16S/19S intron from Simian Virus 40), the HSV TK gene (described inEuropean patent application EP 95202213), the SV40-derivedpolyadenylation signal (nt. 2533–2668 of the SV40 sequence), followed bythe BglII-ScaI fragment of Ad5 (nt. 3328–6092 of the Ad5 sequence).These fragments are present in a pMLP10-derived (Levrero et al., 1991)backbone. To generate plasmid pAD-CMVhc-TK, plasmid pCMV.TK was digestedwith ClaI (the unique ClaI-site is located just upstream of the TK openreading frame) and dephosphorylated with Calf-Intestine AlkalinePhosphate. To generate a hairpin structure, the syntheticoligonucleotides HP/claI (SEQ ID NO:19) and HP/cla2 (SEQ ID NO:20) wereannealed and phosphorylated on their 5-OH groups with T4-polynucleotidekinase and ATP. The double-stranded oligonucleotide was ligated with thelinearized vector fragment and used to transform E. coli strain “Sure.”Insertion of the oligonucleotide into the ClaI site will disrupt theClaI recognition sites. The oligonucleotide contains a new ClaI sitenear one of its termini. In selected clones, the orientation and theintegrity of the inserted oligonucleotide was verified by sequenceanalyses. A clone containing the oligonucleotide in the correctorientation (the ClaI site at the ITR side) was denoted pAd-CMV-hcTK.This plasmid was co-transfected with ClaI-digested wild-type Adenovirustype 5 DNA into 911 cells. A recombinant adenovirus in which theCMV-hcTK expression cassette replaces the E1 sequences was isolated andpropagated using standard procedures.

To study whether the hairpin can be used as a primer for reverse strandsynthesis on the displaced strand after replication has started at theITR, the plasmid pICLhac is introduced into 911 cells (human embryonicretinoblasts transformed with the adenovirus E1 region). The plasmidpICLhaw serves as a control, which contains the oligonucleotide pairHP/asp1 (SEQ ID NO:17) and HP/asp2 (SEQ ID NO:18) in the reverseorientation but is further completely identical to plasmid pICLhac. Alsoincluded in these studies are plasmids pICLI and pICL (SEQ ID NO:21). Inthe plasmid pICLI, the hairpin is replaced by an adenovirus ITR. PlasmidpICL (SEQ ID NO:21) contains neither a hairpin nor an ITR sequence.These plasmids serve as controls to determine the efficiency ofreplication by virtue of the terminal-hairpin structure. To provide theviral products other than the E1 proteins (these are produced by the 911cells) required for DNA replication, the cultures are infected with thevirus IG.Ad.MLPI.TK after transfection. Several parameters are beingstudied to demonstrate proper replication of the transfected DNAmolecules. First, DNA extracted from the cell cultures transfected withthe aforementioned plasmids and infected with IG.Ad.MLPI.TK virus isbeing analyzed by Southern blotting for the presence of the expectedreplication intermediates, as well as for the presence of the duplicatedgenomes. Furthermore, virus is isolated from the transfected andIG.Ad.MLPI.TK infected cell populations that is capable of transferringand expressing a luciferase marker gene into luciferase negative cells.

Plasmid DNA of plasmids pICLhac, pICLhaw, pICLI, and pICL (SEQ ID NO:21)have been digested with restriction endonuclease SalI and treated withmung bean nuclease to remove the 4 nucleotide single-stranded extensionof the resulting DNA fragment. In this manner, a natural adenovirus 5′ITR terminus on the DNA fragment is created. Subsequently, both thepICLhac and pICLhaw plasmids were digested with restriction endonucleaseAsp718 to generate the terminus capable of forming a hairpin structure.The digested plasmids are introduced into 911 cells, using the standardcalcium phosphate co-precipitation technique, four dishes for eachplasmid. During the transfection, for each plasmid two of the culturesare infected with the IG.Ad.MLPI.TK virus using 5 infectiousIG.Ad.MLPI.TK particles per cell. At twenty hours post-transfection andforty hours post-transfection, one Ad.TK-virus-infected culture and oneuninfected culture are used to isolate small molecular-weight DNA usingthe procedure devised by Hirt. Aliquots of isolated DNA are used forSouthern analysis. After digestion of the samples with restrictionendonuclease EcoRI using the luciferase gene as a probe, a hybridizingfragment of approximately 2.6kb is detected only in the samples from theadenovirus-infected cells transfected with plasmid pICLhac. The size ofthis fragment is consistent with the anticipated duplication of theluciferase marker gene. This supports the conclusion that the insertedhairpin is capable of serving as a primer for reverse strand synthesis.The hybridizing fragment is absent if the IG.Ad.MLPI.TK virus is omittedor if the hairpin oligonucleotide has been inserted in the reverseorientation.

The restriction endonuclease DpnI recognizes the tetranucleotidesequence 5′-GATC-3′ but cleaves only methylated DNA (that is, onlyplasmid DNA propagated in, and derived from, E. coli, not DNA that hasbeen replicated in mammalian cells). The restriction endonuclease MboIrecognizes the same sequences, but cleaves only unmethylated DNA (viz.DNA propagated in mammalian cells). DNA samples isolated from thetransfected cells are incubated with MboI and DpnI and analyzed withSouthern blots. These results demonstrate that only in the cellstransfected with the pICLhac and the pICLI plasmids are largeDpnI-resistant fragments present, which are absent in the MboI treatedsamples. These data demonstrate that only after transfection of plasmidspICLI and pICLhac does replication and duplication of the fragmentsoccur.

These data demonstrate that in adenovirus-infected cells, linear DNAfragments that have on one terminus an adenovirus-derived ITR and at theother terminus a nucleotide sequence that can anneal to sequences on thesame strand when present in single-stranded form, thereby generate ahairpin structure and will be converted to structures that have invertedterminal repeat sequences on both ends. The resulting DNA molecules willreplicate by the same mechanism as the wild-type adenovirus genomes.

Experiment Series 2.

The following demonstrates that the DNA molecules that contain aluciferase marker gene, a single copy of the ITR, the encapsidationsignal, and a synthetic DNA sequence that is capable of forming ahairpin structure are sufficient to generate DNA molecules that can beencapsidated into virions.

To demonstrate that the above DNA molecules containing two copies of theCMV-luc marker gene can be encapsidated into virions, virus is harvestedfrom the remaining two cultures via three cycles of freeze-thaw crushingand is used to infect murine fibroblasts. Forty-eight hours afterinfection, the infected cells are assayed for luciferase activity. Toexclude the possibility that the luciferase activity has been induced bytransfer of free DNA, rather than via virus particles, virus stocks aretreated with DNaseI to remove DNA contaminants. Furthermore, as anadditional control, aliquots of the virus stocks are incubated for 60minutes at 56° C. The heat treatment will not affect the contaminatingDNA but will inactivate the viruses. Significant luciferase activity isonly found in the cells after infection with the virus stocks derivedfrom IG.Ad.MLPI.TK-infected cells transfected with the pICLhac and pICLIplasmids. In neither the noninfected cells nor the infected cellstransfected with the pICLhaw and pICL (SEQ ID NO:21) can significantluciferase activity be demonstrated. Heat inactivation, but not DNaseItreatment, completely eliminates luciferase expression, demonstratingthat adenovirus particles, and not free (contaminating) DNA fragments,are responsible for transfer of the luciferase reporter gene.

These results demonstrate that these small viral genomes can beencapsidated into adenovirus particles and suggest that the ITR and theencapsidation signal are sufficient for encapsidation of linear DNAfragments into adenovirus particles. These adenovirus particles can beused for efficient gene transfer. When introduced into cells thatcontain and express at least part of the adenovirus genes (viz. E1, E2,E4, L, and VA), recombinant DNA molecules that consist of at least oneITR, at least part of the encapsidation signal, and a synthetic DNAsequence that is capable of forming a hairpin structure have theintrinsic capacity to autonomously generate recombinant genomes that canbe encapsidated into virions. Such genomes and vector system can be usedfor gene transfer.

Experiment Series 3.

The following demonstrates that DNA molecules that contain nucleotides3510–35953 (viz. 9.7–100 map units) of the adenovirus type 5 genome(thus lacking the E1 protein-coding regions, the right-hand ITR, and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion of the same strand of the DNA molecule whenpresent in single-stranded form other than the ITR, and as a result iscapable of forming a hairpin structure, can replicate in 911 cells.

In order to develop a replicating DNA molecule that can provide theadenovirus products required to allow the above-mentioned pICLhac vectorgenome and alike minimal adenovectors to be encapsidated into adenovirusparticles by helper cells, the Ad-CMV-hcTK adenoviral vector has beendeveloped. Between the CMV enhancer/promoter region and the thymidinekinase gene, the annealed oligonucleotide pair HP/claI (SEQ ID NO:19)and 2 (SEQ ID NO:20) is inserted. The vector Ad-CMV-hcTK can bepropagated and produced in 911 cells using standard procedures. Thisvector is grown and propagated exclusively as a source of DNA used fortransfection. DNA of the adenovirus Ad-CMV-hcTK is isolated from virusparticles that had been purified using CsC1 density-gradientcentrifugation by standard techniques. The virus DNA has been digestedwith restriction endonuclease ClaI. The digested DNA issize-fractionated on a 0.7% agarose gel, and the large fragment isisolated and used for further experiments. Cultures of 911 cells aretransfected with the large ClaI-fragment of the Ad-CMV-hcTK DNA usingthe standard calcium phosphate coprecipitation technique. Much like inthe previous experiments with plasmid pICLhac, the AD-CMV-hc willreplicate starting at the right-hand ITR. Once the one strand isdisplaced, a hairpin can be formed at the left-hand terminus of thefragment. This facilitates the DNA polymerase to elongate the chaintowards the right-hand side. The process will proceed until thedisplaced strand is completely converted to its double-stranded form.Finally, the right-hand ITR will be recreated, and, in this location,the normal adenovirus replication-initiation and elongation will occur.Note that the polymerase will read through the hairpin, therebyduplicating the molecule. The input DNA molecule of 33250 bp, which hadon one side an adenovirus ITR sequence and at the other side a DNAsequence that had the capacity to form a hairpin structure, has now beenduplicated in a way that both ends contain an ITR sequence. Theresulting DNA molecule will consist of a palindromic structure ofapproximately 66,500 bp.

This structure can be detected in low-molecular-weight DNA extractedfrom the transfected cells using Southern analysis. The palindromicnature of the DNA fragment can be demonstrated by digestion of thelow-molecular-weight DNA with suitable restriction endonucleases andSouthern blotting with the HSV-TK gene as the probe. This molecule canreplicate itself in the transfected cells by virtue of the adenovirusgene products that are present in the cells. In part, the adenovirusgenes are expressed from templates that are integrated in the genome ofthe target cells (viz. the E1 gene products); the other genes residingin the replicating DNA fragment itself. Note, however, that this linearDNA fragment cannot be encapsidated into virions. Not only does it lackall the DNA sequences required for encapsidation, but its size is alsomuch too large to be encapsidated.

Experiment Series 4.

The following demonstrates that DNA molecules that contain nucleotides3503–35953 (viz. 9.7–100 map units) of the adenovirus type 5 genome(thus lacking the E1 protein-coding regions, the right-hand ITR, and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion of the same strand of the DNA molecule otherthan the ITR, and as a result is capable of forming a hairpin structure,can replicate in 911 cells and can provide the helper functions requiredto encapsidate the pICLI- and pICLhac-derived DNA fragments.

The following series of experiments aims to demonstrate that the DNAmolecule described in Experiment Series 3 could be used to encapsidatethe minimal adenovectors described in Experiment Series 1 and 2.

In the experiments, the large fragment isolated after endonucleaseClaI-digestion of Ad-CMV-hcTK DNA is introduced into 911 cells (inconformity with the experiments described in part 1.3) together withendonuclease SalI, mung bean nuclease, endonuclease Asp718-treatedplasmid pICLhac, or, as a control, similarly treated plasmid pICLhaw.After 48 hours, virus is isolated by freeze-thaw crushing of thetransfected cell population. The virus preparation is treated withDNaseI to remove contaminating-free DNA. The virus is used subsequentlyto infect Rat2 fibroblasts. Forty-eight hours post-infection, the cellsare assayed for luciferase activity. Significant luciferase activity canbe demonstrated only in the cells infected with virus isolated from thecells transfected with the pICLhac plasmid and not with the pICLhawplasmid. Heat inactivation of the virus prior to infection completelyabolishes the luciferase activity, indicating that the luciferase geneis transferred by a viral particle. Infection of 911 cells with thevirus stock did not result in any cytopathological effects,demonstrating that the pICLhac is produced without any infectious helpervirus that can be propagated on 911 cells. These results demonstratethat the proposed method can be used to produce stocks of minimaladenoviral vectors that are completely devoid of infectious helperviruses and are able to replicate autonomously on adenovirus-transformedhuman cells or on nonadenovirus-transformed human cells.

Besides the system described in this application, another approach forthe generation of minimal adenovirus vectors has been disclosed inInternational Patent Publication WO 94/12649. The method described in WO94/12649 exploits the function of the protein IX for the packaging ofminimal adenovirus vectors (Pseudo Adenoviral Vectors (“PAV”) in theterminology of WO 94/12649). PAVs are produced by cloning an expressionplasmid with the gene of interest between the left-hand (including thesequences required for encapsidation) and the right-hand adenoviralITRs. The PAV is propagated in the presence of a helper virus.Encapsidation of the PAV is preferred compared with the helper virusbecause the helper virus is partially defective for packaging (either byvirtue of mutations in the packaging signal or by virtue of itssize-virus genomes greater than 37.5 kb package inefficiently). Inaddition, the authors propose that, in the absence of the protein IXgene, the PAV will be preferentially packaged. However, neither of thesemechanisms appear to be sufficiently restrictive to allow packaging ofonly PAVs/minimal vectors. The mutations proposed in the packagingsignal diminish packaging but do not provide an absolute block, as thesame packaging activity is required to propagate the helper virus. Also,neither an increase in the size of the helper virus nor the mutation ofthe protein IX gene will ensure that PAV is packaged exclusively. Thus,the method described in WO 94/12649 is unlikely to be useful for theproduction of helper-free stocks of minimal adenovirus vectors/PAVs.

Although the application has been described with reference to certainpreferred embodiments and illustrative examples, the scope of theinvention is to be determined by reference to the appended claims.

REFERENCES

-   Berk A J (1986): Ann. Rev. Genet. 20, 45–79.-   Bernards R, Schrier P I, Bos J L, and Eb A J Vd (1983): Role of    adenovirus types 5 and 12 early region 1b tumor antigens in    oncogenic transformation. J. Virology 127, 45–53.-   Bett A J, Prevec L, and Graham F L (1993): Packaging Capacity and    Stability of Human Adenovirus Type-5 Vectors. J. Virology 67,    5911–5921.-   Blaese M, Blankenstein T, Brenner M, Cohen-Hageenauer O, Gansbacher    B, Russell S, Sorrentino B, and Velu T (1995). Vectors in cancer    therapy: how will they deliver? Cancer Gene Therapy 2, 291–297.-   Boshart M, Weber F, Jahn G, Dorsch-Häler K, Fleckenstein B, and    Scaffner W (1985): A very strong enhancer is located upstream of an    immediate early gene of human Cytomegalovirus. Cell 41, 521–530.-   Bout A, Imler J L, Schulz H, Perricaudet M, Zurcher C, Herbrink P,    Valerio D, and Pavirani A (1994a): In vivo adenovirus-mediated    transfer of human CFTR cDNA to Rhesus monkey airway epithelium:    efficacy, toxicity and safety. Gene Therapy 1, 385–394.-   Bout A, Perricaudet M, Baskin G, Imler J L, Scholte B J, Pavirani A,    and Valerio D (1994b): Lung gene therapy: in vivo adenovirus    mediated gene transfer to rhesus monkey airway epithelium. Human    Gene Therapy 5, 3–10.-   Brody S L, and Crystal R G (1994): Adenovirus-mediated in vivo gene    transfer. Ann. N. Y. Acad. Sci. 716, 90–101.-   Brough D E, Cleghon V, and Klessig D F (1992): Construction,    characterization, and utilization of cell lines which inducibly    express the adenovirus DNA-binding protein. J. Virology 190(2),    624–34.-   Brough D E, Rice S A, Sell S, and Klessig D F (1985): Restricted    changes in the adenovirus DNA-binding protein that lead to extended    host range or temperature-sensitive phenotypes. J. Virology 55,    206–212.-   Daniell E (1976): Genome structure of incomplete particles of    adenovirus. J. Virology 19, 685–708.-   Elsen P J Vd, Houweling A, and Eb A J Vd (1983). Expression of    region E1B of human adenoviruses in the absence of region E1A is not    sufficient for complete transformation. J. Virology 128, 377–390.-   Engelhardt J F, Litzky L, and Wilson J M (1994a): Prolonged    transgene expression in cotton rat lung with recombinant    adenoviruses defective in E2A. Human Gene Therapy 5. 1217–1229.-   Engelhardt J F, Simon R H, Yang Y, Zepeda M, Weber-Pendleton S,    Doranz B, Grossman M, and Wilson J M (1993): Adenovirus-mediated    transfer of the CFTR gene to lung or nonhuman primates: biological    efficacy study. Human Gene Therapy 4, 759–769.-   Engelhardt J F, Ye X, Doranz B, and Wilson J M (1994b): Ablation of    E2A in recombinant adenoviruses improves transgene persistence and    decreases inflammatory response in mouse liver. Proc. Nat'Acad. Sci.    USA 91, 6196–200.-   Fang B, Wang H, Gordon G, Bellinger D A, Read M S, Brinkhous K M,    Woo S L C, and Eisensmith R C (1996): Lack of persistence of    E1-recombinant adenoviral vectors containing a temperature sensitive    E2A mutation in immunocompetent mice and hemophilia dogs. Gene    Therapy 3, 217–222.-   Fallaux F J, Kranenburg O, Cramer S J, Houweling A, Ormondt Hv,    Hoeben R C, and Eb A J Vd (1996): Characterization of 911: a new    helper cell line for the titration and propagation of    early-region-1-deleted adenoviral vectors. Human Gene Therapy 7,    215–222.-   Gooding L R, Aquino L, Duerksen-Hughes P J, Day D, Horton T M, Yei    S, and Wold W S M (1991): The E1B 19,000-molecular-weight protein of    group C adenoviruses prevents tumor necrosis factor cytolysis of    human cells but not of mouse cells. J. Virology 65, 3083–3094.-   Gräble M, and Hearing P (1990): adenovirus type 5 packaging domain    is composed of a repeated element that is functionally redundant. J.    Virolgy 64, 2047–2056.-   Gräble M, and Hearing P (1992): cis and trans requirements for the    selective packaging of adenovirus type-5 DNA. J. Virology 66,    723–731.-   Graham F L, and Eb AJVd (1973): A new technique for the assay of    infectivity of human adenovirus 5 DNA. J. Virology 52, 456–467.-   Graham F L, Smiley J, Russell W C, and Naira R (1977):    Characteristics of a human cell line transformed by DNA from    adenovirus type 5. J. Gen. Virol. 36, 59–72.-   Haddada H, Ragot T, Cordier L, Duffour M T, and Perricaudet M    (1993): Adenoviral interleukin-2 gene transfer into P815 tumor cells    abrogates tumorigenicity and induces antitumoral immunity in mice.    Human Gene Therapy 4, 703–11.-   Hay R T, Stow N D, and McDougall I M (1984): Replication of    adenovirus minichromosomes. J. Mol. Biol. 174, 493–510.-   Hearing P, Samulski R J, Wishart W L, and Shenk T (1987):    Identification of a repeated sequence element required for efficient    encapsidation of the adenovirus type 5 chromosome. J. Virology 61,    2555–2558.-   Horwitz M S (1990): Adenoviridae and their replication, pp.    1679–1740. In B. N. Fields, and D. M. Knipe (Eds): Virology, Raven    Press, Ltd., New York.-   Hu C H, Xu F Y, Wang K, Pearson A N, and Pearson G D (1992):    Symmetrical adenovirus minichromosomes have hairpin replication    intermediates. Gene 110, 145–150.-   Imler J L, Chartier C, Dreyer D, Dieterle A, Sainte-Marie M, Faure    T, Pavirani A, and Mehtali M (1996): Novel complementation cell    lines derived from human lung carcinoma A549 cells support the    growth of E1-deleted adenovirus vectors. Gene Therapy 3, 75–84.-   Jochemsen A G, Peltenburg L T C, Pas M F W T, Wit C Md, Bos J L, and    Eb A J Vd (1987): EMBO J. 6, 3399–3405.-   Klessig D F, and Grodzicker T (1979): Mutations that allow human Ad2    and Ad5 to express late genes in monkey cell maps in the viral gene    encoding the 72K DNA-binding protein. Cell 17, 957–566.-   Klessig D, Grodzicker T, and Cleghon V (1984): Construction of human    cell lines which contain and express the adenovirus DNA binding    protein gene by co-transformation with the HSV-1 tk gene. Virus Res.    1, 169–188.-   Kruijer W, Nicolas J C, Schaik F Mv, and Sussenbach J S (1983):    Structure and function of DNA binding proteins from revertants of    adenovirus type 5 mutants with a temperature-sensitive DNA    replication. Virology 124, 425–433.-   Lechner R L, and Kelly Jr. T J (1977): The structure of replicating    adenovirus 2 DNA molecules. J. Mol. Biol. 174, 493–510.-   Leij L d, Postmus P E, Buys CHCM, Elema J D, Ramaekers F, Poppema S,    Brouwer M, Veen A Y vd, Mesander G, and The TH (1985):    Characterization of three new variant type cell lines derived from    small cell carcinoma of the lung. Cancer Res. 45, 6024–6033.-   Levrero M, Barban V, Manteca S, Ballay A, Balsamo C, Avantaggiati M    L, Natoli G, Skellekens H, Tiollais P, and Perricaudet M (1991):    Defective and nondefective adenovirus vectors for expressing foreign    genes in vitro and in vivo. Gene 101, 195–202.-   Lochmüller H, Jani A, Huard J, Prescott S, Simoneau M, Massie B,    Karpati G, and Acasdi G (1994): Emergence of early region    1-containing replication-competent adenovirus in stocks of    replication-defective adenovirus recombinants (ΔE1–ΔE3) during    multiple passages in 293 cells. Human Gene Therapy 5, 1485–1492.-   Matsui T, Murayama M, and Mita T (1986): Adenovirus 2 peptide IX is    expressed only on replicated DNA molecules. Mol. Cell Biol. 6,    4149–4154.-   Michelson, A M, Markham A F, and Orkin S H (1983): Isolation and DNA    sequence of a full-length cDNA clone for human X-chromosome encoded    phosphoglycerate kinase. Proc. Nat'l Acad. Sci. USA 80, 472–476.-   Morin J E, Lubeck M D, Barton J E, Conley A J, Davis A R, and Hung P    P (1987): Recombinant adenovirus induces antibody response to    hepatitis B virus surface antigens. Proc. Nat'l Acad. Sci. USA, 84,    4626–4630.-   Nicolas J C, Suarez F, Levine A J, and Girard M (1981):    Temperature-independent revertants of adenovirus H5ts125 and H5ts107    mutants in the DNA binding protein: isolation of a new class of host    range temperature conditional revertants. J. Virology 108, 521–524.-   Ostrove J M (1994): Safety testing programs for gene therapy viral    vectors. Cancer Gene Therapy 1, 125–131.-   Pacini D L, Dubovi E J, and Clyde W A (1984): J. Infect. Dis. 150,    92–97.-   Postmus P E, Ley L d, Veen AYvd, Mesander G, Buys CHCM, and Elema J    D (1988): Two small cell lung cancer cell lines established from    rigid bronchoscope biopsies. Eur. J. Clin. Oncol. 24, 753–763.-   Rice S A, and Kiessig D F (1985): Isolation and analysis of    adenovirus type 5 mutants containing deletions in the gene encoding    the DNA-binding protein. J. Virology 56, 767–778.-   Roberts B E, Miller J S, Kimelman D, Cepko C L, Lemischka I R, and    Mulligan R C (1985): J. Virology 56, 404–413.-   Shapiro D L, Nardone L L, Rooney S A, Motoyama E K, and Munoz J L    (1978): Phospholipid biosynthesis and secretion by a cell line    (A549) which resembles type II alveolar epithelial cells. Biochim.    Biophys. Acta 530, 197–207.-   Simon R H, Engelhardt J F, Yang Y, Zepeda M, Weber-Pendleton S,    Grossman M, and Wilson J M (1993): Adenovirus-mediated transfer of    the CFTR gene to lung of nonhuman primates: toxicity study. Human    Gene Therapy 4, 771–780.-   Singer-Sam J, Keith D H, Tani K, Simmer R L, Shively L, Lindsay S,    Yoshida A, and Riggs A D (1984): Sequence of the promoter region of    the gene for X-linked 3-phosphoglycerate kinase. Gene 32, 409–417.-   Stein R W, and Whelan J (1989): Insulin gene enhancer activity is    inhibited by adenovirus 5 E1A gene products. Mol Cell Biol 9,    4531–4.-   Stratford-Perricaudet L D, and Perricaudet M (1991): Gene transfer    into animals: the promise of adenovirus, pp. 51–61. In O.    Cohen-Adenauer, and M. Boiron (Eds): Human Gene Transfer, John    Libbey Eurotext.-   Telling G C, Perera S, Szatkowski O M, and Williams J (1994):    Absence of an essential regulatory influence of the adenovirus E1B    19-kilodalton protein on viral growth and early gene expression in    human diploid WI38, HeLa, and A549 cells. J. Virology 68, 541–7.-   Tooze J (1981): DNA Tumor Viruses (revised). Cold Spring Harbor    Laboratory. Cold Spring Harbor, N.Y.-   Vieira J, and Messing J (1987): Production of single stranded    plasmid DNA, pp. 3–11: Methods in Enzymology, Acad. Press Inc.-   Vincent A J P E, Esandi Md C, Someren GDv, Noteboom J L, Avezaat C J    J, Vecht C, Smitt PAES, Bekkum D Wv, Valerio D, Hoogerbrugge P M,    and Bout A (1996a): Treatment of Lepto-meningeal metastasis in a rat    model using a recombinant adenovirus containing the HSV-tk gene. J.    Neurosurg. in press.-   Vincent A J P E, Vogels R, Someren Gv, Esandi Md C, Noteboom J L,    Avezaat C J J, Vecht C, Bekkum D Wv, Valerio D, Bout A, and    Hoogerbrugge P M (1996b): Herpes Simplex Virus Thymidine Kinase gene    therapy for rat malignant brain tumors. Human Gene Therapy 7,    197–205.-   Wang K, and Pearson G D (1985): Adenovirus sequences required for    replication in vivo. Nucl. Acids Res. 13, 5173–5187.-   White E, Denton A, and Stillman B (1988): J. Virolgy 62, 3445–3454.-   Yang Y, Li Q, Ertl H C J, and Wilson J M (1995): Cellular and    humoral immune responses viral antigens create barriers to    lung-directed gene therapy with recombinant adenoviruses. J. Virolgy    69, 2004–2015.-   Yang Y, Nunes F A, Berencsi K, Furth E E, Gonczol E, and Wilson J M    (1 994a): Cellular immunity to viral antigens limits E1-deleted    adenoviruses for gene therapy. Proc Nat'l Acad. Sci. USA 91,    4407–11.-   Yang Y, Nunes F A, Berencsi K, Gonczol E, Engelhardt J F, and Wilson    J M (1994b): Inactivation of E2A in recombinant adenoviruses    improves the prospect for gene therapy in cystic fibrosis. Nature    Genetics 7, 362–9.-   Zantema A, Fransen J A M, Davis-Olivier A, Ramaekers F C S, Vooijs G    P, Deleys B, and Eb A J Vd (1985): Localization of the E1B proteins    of adenovirus 5 in transformed cells, as revealed by interaction    with monoclonal antibodies. J. Virology 142, 44–58.

1. A method of producing a recombinant nucleic acid molecule, saidmethod comprising: providing a complementing cell comprising at least anadenoviral EIA gene; introducing a precursor molecule into saidcomplementing cell, wherein said precursor molecule is a nucleic acidmolecule based on or derived from an adenovirus, said precursor moleculehas one functional inverted terminal repeat, said precursor moleculecomprises all other adenovirus derived genetic information necessary forreplication not present in said complementing cell, and said precursormolecule is in a linear and essentially single stranded form andcomprises, at the precursor molecule's 3′ terminus, a recombinantlyfused sequence complementary to an upstream part of the same strand ofthe precursor molecule, to allow said recombinantly fused sequence andsaid upstream part to form base pairs and function as a start-site for anucleic acid polymerase; and producing a recombinant nucleic acidmolecule by the action of said nucleic acid polymerase on said precursormolecule in said complementing cell.
 2. The method according to claim 1,wherein said precursor molecule comprises a nucleic acid having anadenovirus hr400–404 mutation.
 3. The method according to claim 1,wherein said precursor molecule comprises an adenovirus E2A ts125mutation.
 4. The method according to claim 1, wherein said precursormolecule comprises an adenovirus E2 region under the control of aninducible promoter.
 5. The method according to claim 1, wherein saidprecursor molecule lacks overlapping sequences with the nucleic acid ofthe complementing cell into which it is introduced, said overlappingsequences otherwise enabling homologous recombination leading toreplication competent virus in the complementing cell.
 6. The methodaccording to claim 1, wherein said precursor molecule lacks a functionalencapsidation signal.
 7. The method according to claim 1, wherein saidcomplementing cell further comprises an E1B gene of an adenovirus. 8.The method according to claim 1, wherein said complementing cell is aPER.C6 cell as deposited under number ECACC
 96022940. 9. The methodaccording to claim 1, wherein producing a recombinant nucleic acidmolecule by the action of said nucleic acid polymerase on said precursormolecule in said complementing cell results in a recombinant nucleicacid molecule greater than 38 kb in length.
 10. A recombinant nucleicacid molecule produced by a process comprising: providing acomplementing cell comprising at least an adenoviral EIA gene;introducing a precursor molecule into said complementing cell, wherein:said precursor molecule is a nucleic acid molecule based on or derivedfrom an adenovirus; said precursor molecule has one functional invertedterminal repeat; said precursor molecule comprises all other adenovirusderived genetic information necessary for replication not present insaid complementing cell; and said precursor molecule is in a linear andessentially single stranded form and comprises, at the precursormolecule's 3′ terminus, a recombinantly fused sequence complementary toan upstream part of the same strand of the precursor molecule, to allowsaid recombinantly fused sequence and said upstream part to form basepairs and function as a start-site for a nucleic acid polymerase; andproducing the recombinant nucleic acid molecule by the action of anucleic acid polymerase in said complementing cell on said precursormolecule.
 11. The recombinant nucleic acid molecule produced by theprocess of claim 10, wherein producing the recombinant nucleic acidmolecule by the action of a nucleic acid polymerase in saidcomplementing cell on said precursor molecule results in the recombinantnucleic acid molecule being greater than 38 kb in length.
 12. Therecombinant nucleic acid molecule produced by the process of claim 11,wherein said recombinant nucleic acid molecule lacks a functionalencapsidation signal.
 13. The recombinant nucleic acid molecule producedby the process of claim 10, wherein said recombinant nucleic acidmolecule further comprises an adenovirus hr400–404 mutation.
 14. Therecombinant nucleic acid molecule produced by the process of claim 10,wherein said recombinant nucleic acid molecule comprises an adenovirusE2A ts125 mutation.
 15. The recombinant nucleic acid molecule producedby the process of claim 10, wherein said recombinant nucleic acidmolecule comprises an adenovirus E2 region under the control of aninducible promoter.
 16. The recombinant nucleic acid molecule producedby the process of claim 10, wherein said recombinant nucleic acidmolecule lacks overlapping sequences with a nucleic acid of thecomplementing cell into which it is transferred, said overlappingsequences otherwise enabling homologous recombination resulting inproducing replication competent virus in the complementing cell.
 17. Therecombinant nucleic acid molecule produced by the process of claim 10,wherein said recombinant nucleic acid molecule lacks a functionalencapsidation signal.
 18. A cell comprising the recombinant nucleic acidmolecule produced by the process of claim
 10. 19. A method ofpropagating a helper-dependent adenoviral vector in a complementingcell, said method comprising: providing the recombinant nucleic acidmolecule produced by the process of claim 10 to a complementing cell;introducing a helper-dependent adenoviral vector into said complementingcell; and propagating said helper-dependent adenoviral vector in saidcomplementing cell.
 20. The method according to claim 19, wherein saidhelper dependent adenoviral vector lacks overlapping sequences with saidrecombinant nucleic acid molecule, said overlapping sequences otherwiseenabling homologous recombination leading to replication competent virusin said cell.
 21. The method according to claim 20, wherein therecombinant nucleic acid molecule comprises a packaging defectiverecombinant nucleic acid molecule greater than 38 kb in length.
 22. Themethod according to claim 20, wherein the recombinant nucleic acidmolecule comprises a packaging defective recombinant nucleic acidmolecule lacking a functional encapsidation signal.
 23. The methodaccording to claim 21, wherein the packaging defective recombinantnucleic acid molecule lacks a functional encapsidation signal.